Array-based methods for analysing mixed samples using differently labelled allele-specific probes

ABSTRACT

This disclosure provides methods and kits useful in analysis of mixed nucleic acid populations, including for multiplex genotyping of a mixed nucleic acid sample and for detecting differences in copy number of a target polynucleotide and/or a target chromosome (e.g., microdeletions, duplications and aneuploidies). The disclosure also provides methods and systems useful in the diagnosis of genetic abnormalities in a mixed nucleic acid population taken non-invasively from an organism, such as a sample of blood, plasma, serum, urine stool or saliva. The disclosed methods and systems find use in multiple applications, including prenatal testing and cancer diagnostics. The method is based on the hybridisation of amplified fragments obtained from the sample, e.g. using molecular inversion probes (MIP) to an oligonucleotide array and the detection of the alleles based on different signals from the different alleles of the SNP.

RELATED APPLICATIONS

This application is a U.S. 371 application of International ApplicationNo. PCT/US2018/035684, filed Jun. 1, 2018, which claims priority fromU.S. Provisional patent application No. 62/514,629, filed Jun. 2, 2017,which is hereby incorporated herein by reference in its entirety for allpurposes.

TECHNICAL FIELD

This disclosure provides methods and systems useful in array-basedanalysis of mixed nucleic acid populations, including for genotyping andcopy number analysis of the various subpopulations of the mixed nucleicacid population. The disclosure also provides methods and systems usefulin the diagnosis of genetic abnormalities in a mixed nucleic acidpopulation taken from an organism. For example, disclosed herein aremethods and systems useful in the diagnosis of fetal geneticabnormalities or tumor genetic abnormalities using samples obtainednoninvasively from pregnant females or patients. Such samples caninclude mixed nucleic acid populations derived from blood, plasma,serum, urine, stool or saliva.

BACKGROUND

Analysis of mixed nucleic acid populations, for example DNA and RNAsamples obtained from a single tissue source such as blood, urine orsaliva but containing distinct nucleic acid subpopulations, has elicitedsignificant interest in the research and health care communities. Usingsuitable methods, mixed nucleic acid populations derived from cell-freeDNA (or RNA) taken from pregnant females can be analyzed to determinefetal characteristics, including disease inheritance. Similarly, mixednucleic acid populations derived from cell-free DNA (or RNA) taken fromcancer patients can be analyzed to determine various characteristicssuch as tumor malignancy, tumor origin or drug susceptibility. Whileanalysis of such mixed nucleic acid populations can be technicallycomplex due to the high degree of similarity between the varioussubpopulations, the difficulty of the analysis is outweighed by the easeof obtaining appropriate nucleic acid samples cheaply, quickly andnon-invasively through procedures such as phlebotomy or urine/salivacollection. One mode of analyzing cell-free DNA, nucleic acidsequencing, is informative but costly on a per-sample and time-consumingMicroarray analysis is cheaper and quicker than sequencing, but currentcommercial embodiments of microarray products do not readily supportdiscrimination between the different and highly similar subpopulationspresent in a mixed nucleic acid population. As a result of the lowconcentration of fetal DNA in maternal samples, and low concentration oftumor DNA in a blood sample containing circulating tumor cells, singleor low multiplex assays are unlikely to differentiate between ananeuploid fetus (e.g., trisomy of chromosome 21) from a euploid fetus,or a tumor cell from a healthy cell in a cancer patient. For example,fetal DNA can be present at levels of between 4%-15% of total cell-freeDNA in blood; DNA derived from a particular fetal chromosome wouldrepresent one-twenty-third of such fetal DNA. Detection of a trisomywould require reliable detection of signal changes as low as 1-2% abovebackground. Moreover, the analysis is further complicated by the limitedamount of nucleic acid available through non-invasive sampling methods.For example, a maternal sample of 10 mls of whole blood can yieldbetween 5 and 15 ng of purified cell-free DNA in a typical assay.

Due to the current challenges posed by such non-invasive approaches, amajority of pregnant women are subject to prenatal testing, includingmaternal serum screening and/or an ultrasound test, to determine risksfor common birth defects, such as those resulting from trisomy 13, 18,and 21. However, the sensitivity and specificity of such tests are verypoor leading to high false positive rates. As a result of the high falsepositive rates of such conventional tests, individuals typically mustconduct follow-up testing with an invasive diagnostic test, such asChorionic Villus Sampling (CVS) between 11 and 14 weeks gestation oramniocentesis after 15 weeks gestation. These invasive procedures carrya risk of a miscarriage of around one percent (see Mujezinovic andAlfirevic, Obstet. Gynecol., 110:687-694 (2011)). Current analysis offetal cells typically involves karyotyping or fluorescent in situhybridization (FISH) and does not provide information about single genetraits. As a result, additional tests are required for identification ofsingle gene diseases and disorders. Because prenatal diagnosis can becritical for management of a pregnancy with chromosomal abnormalitiesand localized genetic abnormalities, an accurate and early diagnosis isimportant to allow for interventional care before or during delivery andto prevent devastating consequences for the neonate.

Similarly, on the cancer front, powerful tools such as OncoScan® havebeen developed for purposes of diagnosing cancers. However, such samplesare typically biopsy samples taken in invasive procedures that are bothexpensive and potentially risky to the patient. Through the use ofmicroarray-based technology, researchers are able to identify largenumbers of Single Nucleotide Polymorphisms (SNPs) on a single array,which allows for the rapid and accurate detection of geneticabnormalities in a subject. As an example of one such product is the SNPdetection microarray product from Affymetrix called OncoScan®. TheOncoScan® product provides genome-wide copy number andloss-of-heterozygosity (LOH) profiles from solid tumor samples. Such atechnology is a powerful tool in cancer diagnostics because it helps toovercome significant challenge due to the difficulty of working withlimited amounts of DNA from highly degraded FFPE samples. See, forexample, U.S. Pat. No. 8,190,373. However, such technologies are findingapplication in numerous other fields, as well. Specifically, geneticabnormalities account for a wide number of pathologies, includingpathologies caused by chromosomal aneuploidy (e.g., Down syndrome),germline mutations in specific genes (e.g., sickle cell anemia), andpathologies caused by somatic mutations (e.g., cancer), and in manycases, the detection of such genetic abnormalities is complicated byinvasive diagnostic procedures.

As such, the development of a microarray based test that is sensitiveand specific enough to detect genetic abnormalities in samples of mixednucleic acid populations obtained by non-invasive means with lowfalse-positive and false-negative rates would be of benefit to the fieldof molecular diagnostics. Recently, Ariosa Diagnostics reported studiesinvolving microarray based analysis of cell-free DNA from maternal bloodto detect the presence of fetal aneuploidies. See, e.g., Stokowski etal., Prenatal Diagnosis 35:1243-1246 (2015). Such methods involvedanalysis of bulk signals from non-polymorphic loci (i.e., loci that areexpected to be identical for both mother and fetus) to estimatechromosomal copy number by simply measuring fluctuations in total signaldetected from both maternal and fetal DNA at a given genetic locus. Thisnecessitates a design strategy whereby the array is configured tointerrogate non-polymorphic loci to determine copy number of theunderlying chromosomes. There is a need to develop improved methods (asall as associated compositions, systems, devices and instruments) thatleverages the high-throughput genotyping capabilities ofmicroarray-based analysis to generate data from a single set ofinterrogation sites (for example, a data from a single set ofpolymorphic loci in mixed DNA populations), which can then be used toboth genotype and estimate copy number of a given locus or chromosomewithin the major and minor DNA populations within mixed nucleic acidpopulations.

Described herein are methods and systems for analyzing a mixed nucleicacid sample to detect differences in copy number of a targetpolynucleotide, such as a detection of copy number variants indicatingchromosomal aneuploidy, as well as methods of genotyping such targetpolynucleotides even when present at low levels within a mixed nucleicacid population.

SUMMARY

This Summary is provided to introduce various aspects of the disclosurethat are further described below in the Detailed Description. ThisSummary is not intended to limit the scope of the claimed subjectmatter. Other features, details, utilities, and advantages of theclaimed subject matter will be apparent from the following writtenDetailed Description including those aspects illustrated in theaccompanying drawings and defined in the appended claims.

In one aspect, the disclosure provides methods for analyzing a nucleicacid sample obtained from an organism. The nucleic acid sample caninclude DNA and/or RNA, or synthetic derivatives thereof. The nucleicacid sample can include cell-free DNA and/or cell-free RNA. In someembodiments, the nucleic acid sample includes a mixed nucleic acidpopulation. The nucleic acid sample containing the mixed nucleic acidpopulation can be obtained from a single organism. The mixed nucleicacid population can include nucleic acid of fetal origin and maternalorigin. The mixed nucleic acid population can include nucleic acidoriginating from tumor and normal cells.

The methods described herein can further include obtaining or derivingfrom an organism a nucleic acid sample containing a mixed nucleic acidpopulation. The obtaining or deriving optionally includes any one ormore of the following steps: labeling (including bulk labeling orstochastic labeling), single-molecule labeling, amplification, ligationto other nucleic acid sequences, circularization, hybridization, targetselection, methylation or binding to methylation-specific reagents,antibody binding, target capture, precipitation, elution, and the like,In some embodiments, the mixed nucleic acid sample includes a majorsubpopulation and a minor subpopulation. The major subpopulation isoptionally present at greater than 50% of total nucleic acid in themixed nucleic acid population. The major subpopulation can be present atgreater than 50% of total nucleic acid in the nucleic acid sample. Insome embodiments, the major and minor subpopulations each include atarget sequence located in a first chromosomal region. The targetsequence of the major and minor subpopulations can be the same sequenceor overlapping sequences. In some embodiments, the target sequencecontains a polymorphic site. The polymorphic site can include a sequencecontaining a first nucleotide variant and/or a second nucleotidevariant, optionally at the same site. In some embodiments, thepolymorphic sequence includes a single nucleotide polymorphism (SNP).The SNP can include a single nucleotide whose identity defines anallelic variant of the polymorphic site. The polymorphic site caninclude a major allele or a minor allele or both (e.g., in the case of adiploid organism).

In some embodiments, the methods described herein (as well as relatedcompositions, kits, and systems) involve the selective enrichment ofcertain genetic sequences of interest. The selective enrichment caninclude targeted amplification, which may be performed in singleplex ormultiplex formats. In some embodiments, the described methods caninclude use of target-specific primers or probes. Optionally, themethods include use of a molecular inversion probe. In some embodiments,the methods include hybridizing the primer or probe (e.g., the molecularinversion probe) to a target sequence. Optionally, the primer or probecan be extended in a target-specific manner. In some embodiments, theprobe is a molecular inversion probe that hybridizes adjacent to orupstream of a polymorphic site. The methods can include extending theprimer or probe by incorporating a nucleotide whose identity correspondsto the sequence of one or more polymorphisms in the polymorphic site.

In some embodiments, the methods described herein include genotyping thepolymorphic site. In further embodiments, the genotyping includeshybridizing at least one nucleic acid fragment containing or derivedfrom the nucleic acid population and containing the polymorphic site toan oligonucleotide probe. The oligonucleotide probe can optionally belocated within an array of other probes, or can be hybridized to anotheroligonucleotide probe present in an array.

In some embodiments, the described methods further include detectingfrom the oligonucleotide array, using a detector, a first signalindicating the presence or absence of the first nucleotide variant (“Asignal”). In some embodiments, the described methods include detecting asecond signal indicating the presence or absence of the secondnucleotide variant (“B signal”). In some embodiments, the describedmethods can include detecting both the first signal and the secondsignal from the same array. In some embodiments, the first signal canindicate the present or absence of a first allelic form of thepolymorphic site (“A allele”). The second signal can indicate thepresent or absence of a second allelic form of the polymorphic site (“Ballele”). In some embodiments, the major subpopulation includes the Aallele and the minor subpopulation includes the B allele. In someembodiments, the described methods further include genotyping the majorsubpopulation, the minor subpopulation or both the major and minorsubpopulation, optionally using the A signal, the B signal, or both theA and B signals. In some embodiments, the described methods furtherinclude estimating or calculating the copy number of the target nucleicacid sequence including the polymorphic site in the major subpopulation,the minor subpopulation or both the major and minor subpopulation,optionally using the A signal, the B signal or both the A and B signals.The methods can include calculating the copy number of the firstchromosomal region using the A signal, the B signal or both the A and Bsignals. The methods can include detecting the presence or absence of ananeuploidy. In some embodiments, the methods can include calculating therelative proportions of nucleic acid derived from the major and minorsubpopulations using the A signal, the B signal or both the A and Bsignals. The methods can include calculating the fetal fraction of thenucleic acid sample using the A signal, the B signal or both the A and Bsignals. In some embodiments, the methods can further include any one ormore of the following steps: (a) determining the copy number of thefirst chromosomal region in the minor subpopulation using the firstsignal and the second signal; (b) determining the copy number of thefirst chromosomal region in the major subpopulation using the firstsignal and the second signal; (c) determining the genotype of thepolymorphic site for the minor subpopulation using the first signal andthe second signal; (d) determining the genotype of the polymorphic sitefor the major subpopulation using the first signal and the secondsignal, and (e) further including determining the relative amounts ofthe major subpopulation and the minor subpopulation in the mixed nucleicacid population using the first signal and the second signal.

In another aspect, the disclosure provides methods for determining acopy number variation in a mixed nucleic acid sample obtained from anorganism, the method comprising one or more of the following steps:

a. isolating genomic DNA to form a mixed nucleic acid sample containinga mixed nucleic acid population that includes a major subpopulation anda minor subpopulation;

b. contacting the nucleic acid sample with a pool of linear molecularinversion probes to provide an annealing mixture comprising a pluralityof linear molecular inversion probe-DNA fragment complexes;

c. dividing the annealing mixture into a first channel composition and asecond channel composition;

d. adding a mixture of deoxynucleotides to each of the first and secondchannel composition, wherein the mixture of deoxynucleotides added tothe first channel composition is different from the mixture ofdeoxynucleotides added to the second channel composition;

e. contacting the first and second channel compositions with a ligase toform first and second circularized probe compositions;

f. optionally contacting the first circularized probe composition andthe second circularized probe composition with a first exonuclease tocleave remaining linear molecular inversion probes and nucleic acidfragments;

g. cleaving the first circularized and second probe compositions to formnucleic acid fragments containing or derived from the nucleic acidpopulation;

h. amplifying the first and second nucleic acid fragments containing orderived from the nucleic acid population;

i. combining the first and second nucleic acid fragments containing orderived from the nucleic acid population;

j. digesting the first and second nucleic acid fragments containing orderived from the nucleic acid population;

k. hybridizing at least one nucleic acid fragment containing or derivedfrom the nucleic acid population and containing the polymorphic site toan oligonucleotide probe of an oligonucleotide array;

l. labeling a surface-bound first and second nucleic acid fragmentscontaining or derived from the nucleic acid population with a firstagent that binds to the first nucleotide variant and a second agent thatbinds to the second nucleotide variant; and

m. analyzing the intensity of a signal specific for the first agent andthe intensity of a signal from the second agent to determine a copynumber of a chromosome.

In another aspect, the disclosure provides a kit useful in the detectionof fetal copy number variation comprising:

a. a capture device having a plurality of nucleic acid fragmentscorresponding at least one chromosomal target region attached thereto;

b. a plurality of molecular probes capable of hybridizing to a mixednucleic acid population that includes a major subpopulation and a minorsubpopulation, wherein the major and minor subpopulations each include atarget sequence located in a first chromosomal region and containing apolymorphic site, wherein the polymorphic site can include combinationsof a first nucleotide variant and a second nucleotide variant; and

c. instructions for genotyping and detecting the polymorphic site.

These aspects and other embodiments of the disclosure can be furtherdescribed by the following enumerated clauses:

1. A method for analyzing a mixed nucleic acid sample obtained from anorganism, comprising:

obtaining or deriving from an organism a nucleic acid sample containinga mixed nucleic acid population that includes a major subpopulation anda minor subpopulation, wherein the major and minor subpopulations eachinclude a target sequence located in a first chromosomal region andcontaining a polymorphic site, wherein the polymorphic site can includea first nucleotide variant, a second nucleotide variant or both thefirst and second nucleotide variants;

genotyping the polymorphic site, wherein the genotyping includes: (a)hybridizing at least one nucleic acid fragment containing or derivedfrom the nucleic acid population and containing the polymorphic site toan oligonucleotide probe of an oligonucleotide array; and

(b) detecting from the oligonucleotide array, using a detector, a firstsignal indicating the presence or absence of the first nucleotidevariant (“A signal”) and a second signal indicating the presence orabsence of the second nucleotide variant (“B signal”). Optionally, thefirst nucleotide variant corresponds to a first allelic variant and thesecond nucleotide variant corresponds to a second allelic variant.

2. The method of clause 1, further including determining the copy numberof the first chromosomal region in the minor subpopulation using thefirst signal and the second signal.

3. The method of clause 1, further including determining the copy numberof the first chromosomal region in the major subpopulation using thefirst signal and the second signal.

4. The method of clause 1, further including determining the genotype ofthe polymorphic site for the minor subpopulation using the first signaland the second signal.

5. The method of clause 1, further including determining the genotype ofthe polymorphic site for the major subpopulation using the first signaland the second signal.

6. The method of clause 1, further including determining the relativeamounts of the major subpopulation and the minor subpopulation in themixed nucleic acid population using the first signal and the secondsignal.

7. The method of any of the preceding clauses, wherein the majorsubpopulation and the minor subpopulation originate from differentsources in the organism.

8. The method of any of the preceding clauses, wherein the mixed nucleicacid population includes cell-free DNA.

9. The method of clause 8, wherein the cell-free DNA is obtained orderived from the organism's blood, plasma, serum, urine, stool orsaliva.

10. The method of any of the preceding clauses, wherein the organismincludes a tumor, the major subpopulation includes or is derived fromnormal tissue and the minor subpopulation includes or is derived fromthe tumor.

11. The method of any of the preceding clauses, wherein the organism isa pregnant female, the mixed nucleic acid population is cell-free DNAobtained from the pregnant female's blood, the major subpopulationincludes or is derived maternal nucleic acid and the minor subpopulationincludes or is derived from fetal nucleic acid.

12. The method of clause 11, wherein the minor subpopulation includesfetal DNA present at no greater than 20% of total DNA in the nucleicacid sample.

13. The method of clause 12, wherein the fetal DNA is no greater than15% of total DNA in the nucleic acid sample.

14. The method of clause 12, wherein the fetal DNA is no greater than10% of total DNA in the nucleic acid sample.

15. The method of clause 12, wherein the fetal DNA is no greater than 5%of total DNA in the nucleic acid sample.

16. The method of clause 1, wherein the fetal DNA is no greater than 15%and no less than 1% of total cell-free DNA in the nucleic acid sample.

17. The method of any of the preceding clauses, wherein the mixednucleic acid population contains or is derived from cell-free DNApresent in blood of the organism at concentration of no greater than 5ng/mL and no less than 0.1 ng/mL.

18. The method of clause 1, wherein the amount of mixed nucleic acidpopulation used is no greater than 50 ng, 40 ng, 30 ng, 15 ng, 10 ng, 5ng, 3 ng or 1 ng.

19. The method of clause 1, wherein the polymorphic site includes abi-allelic SNP, the first nucleotide variant is a first allelic variantof the SNP (“A allele”) and the second nucleotide variant is a secondallelic variant of the SNP (“B allele”).

20. The method of any of the preceding clauses, wherein the detectorincludes a first detection channel and a second detection channel, andfurther including the steps of detecting the first signal in the firstdetection channel and the second signal in the second detection channel.

21. The method of clause 19, wherein the SNP can include the A allele orthe B allele, and wherein the SNP genotype can be homozygous for the Aallele (“AA”), homozygous for the B allele (“BB”) or heterozygous(“AB”).

22. The method of any one of the preceding clauses, wherein the step ofgenotyping further includes contacting the nucleic acid sample with apool of linear molecular inversion probes to provide an annealingmixture.

23. The method of clause 22, wherein the pool of linear molecularinversion probes comprises at least 1,000 linear molecular inversionprobes.

24. The method of clause 22, wherein the pool of linear molecularinversion probes comprises at least 5,000 linear molecular inversionprobes.

25. The method of clause 22, wherein the pool of linear molecularinversion probes comprises at least 10,000 linear molecular inversionprobes.

26. The method of clause 22, wherein the pool of linear molecularinversion probes comprises at least 20,000 linear molecular inversionprobes.

27. The method of clause 22, wherein the pool of linear molecularinversion probes comprises less than 200,000 linear molecular inversionprobes.

28. The method of clause 22, wherein the pool of linear molecularinversion probes comprises less than 100,000 linear molecular inversionprobes.

29. The method of clause 22, wherein the pool of linear molecularinversion probes comprises less than 80,000 linear molecular inversionprobes.

30. The method of any one of clauses 22-29, wherein at least 50% of thepool of linear molecular inversion probes binds DNA fragments fromchromosomes 1, 5, 13, 18, 21, X, and Y.

31. The method of any one of clauses 22-29, wherein at least 60% of thepool of linear molecular inversion probes binds DNA fragments fromchromosomes 1, 5, 13, 18, 21, X, and Y.

32. The method of any one of clauses 22-29, wherein at least 70% of thepool of linear molecular inversion probes binds DNA fragments fromchromosomes 1, 5, 13, 18, 21, X, and Y.

33. The method of any one of clauses 22-29, wherein the ratio of thetotal number of linear molecular inversion probes to the total number ofDNA fragment copies is about 40,000:1.

34. The method of any one of clauses 22-29, wherein the ratio of thetotal number of linear molecular inversion probes to the total number ofDNA fragment copies is at least 15,000:1.

35. The method of any one of clauses 22-29, wherein the ratio of thetotal number of linear molecular inversion probes to the total number ofDNA fragment copies is at least 30,000:1.

36. The method of any one of clauses 22-29, wherein the ratio of thetotal number of linear molecular inversion probes to the total number ofDNA fragment copies is less than 100,000:1.

37. The method of any one of clauses 22-29, wherein the ratio of thetotal number of linear molecular inversion probes to the total number ofDNA fragment copies is less than 60,000:1.

38. The method of any one of the preceding clauses, wherein the step ofgenotyping further includes dividing the annealing mixture into a firstchannel composition and a second channel composition.

39. The method of clause 38, wherein the first channel compositioncomprises a mixture of dATP and dTTP.

40. The method of clause 38 or 39, wherein the first channel compositionis substantially free of dGTP or dCTP.

41. The method of any one of clauses 38-40, wherein the second channelcomposition comprises a mixture of dGTP and dCTP.

42. The method of any one of clauses 38-41, wherein the second channelcomposition is substantially free of dATP or dTTP.

43. The method of any one of the preceding clauses, wherein the step ofgenotyping further includes adding a mixture of deoxynucleotides to eachof the first and second channel composition, wherein the mixture ofdeoxynucleotides added to the first channel composition is differentfrom the mixture of deoxynucleotides added to the second channelcomposition.

44. The method of any one of the preceding clauses, wherein the step ofgenotyping further includes contacting the first and second channelcompositions with a ligase to form first and second circularized probecompositions.

45. The method of any one of the preceding clauses, wherein the step ofgenotyping further includes cleaving the first circularized and secondprobe compositions to form nucleic acid fragments containing or derivedfrom the nucleic acid population.

46. The method of any one of the preceding clauses, wherein the step ofgenotyping further includes amplifying the first and second nucleic acidfragments containing or derived from the nucleic acid population.

47. The method of any one of the preceding clauses, wherein the step ofamplifying in carried out in the presence of a polymerase.

48. The method of clause 47, wherein the polymerase is a hot-startpolymerase comprising the polymerase and a polymerase inhibitor.

49. The method of clause 48, wherein the polymerase inhibitor isdisassociated from the polymerase when the temperature is at least 40°C.

50. The method of any one of the preceding clauses, wherein the step ofgenotyping further includes combining the first and second nucleic acidfragments containing or derived from the nucleic acid population.

51. The method of any one of the preceding clauses, wherein the step ofdetecting further includes labeling a surface-bound first and secondnucleic acid fragment containing or derived from the nucleic acidpopulation with a first agent that binds to the first allelic variantand a second agent that binds to the second allelic variant.

52. The method of clause 51, wherein the first agent has an antibody.

53. The method of clause 51 or 52, wherein the first agent has acomplementary sequence to a portion of the first target sequence.

54. The method of any one of clauses 51-53, wherein the first agentfurther comprises a recognition element conjugated to the complementarysequence.

55. The method of clause 54, wherein the recognition element is biotin.

56. The method of any one of clauses 51-55, wherein the first agentfurther comprises a fluorescently labeled avidin.

57. The method of any one of clauses 51-56, wherein the first agentfurther comprises an antibody that binds avidin.

58. The method of clause 57, wherein the antibody that binds avidin islabeled with a biotin.

59. The method of any one of clauses 51-58, wherein the first agentfurther comprises an antibody that binds the recognition element.

60. The method of clause 59, wherein the antibody that binds therecognition element is labeled with a reporter.

61. The method of any one of clauses 51-60, wherein the first agentcomprises a fluorophore.

62. The method of clause 61, wherein the fluorophore of the first agenthas a fluorescence emission peak between about 640 nm and about 680 nm.

63. The method of clause 61 or 62, wherein the fluorophore of the firstagent is allophycocyanin.

64. The method of clause 51, wherein the second agent has acomplementary sequence to a portion of the second target sequence.

65. The method of clause 64, wherein the second agent further has arecognition element conjugated to the complementary sequence.

66. The method of clause 65, wherein the recognition element is FAM.

67. The method of any one of clauses 64-66 wherein the second agentfurther comprises a first antibody that binds the recognition element.

68. The method of any one of clauses 64-66, wherein the second agentfurther comprises a second antibody that binds the first antibody.

69. The method of clause 68, wherein the first antibody, the secondantibody, or both the first and second antibody are labeled with afluorophore.

70. The method of clause 69, wherein the fluorophore of the second agenthas a fluorescence emission peak between about 560 nm and about 600 nm.

71. The method of clause 70, wherein the fluorophore of the second agentis phycoerythrin.

72. The method of any one of the preceding clauses, wherein the step ofcontacting the cell-free DNA composition occurs in reaction volume thatis less than 50 μL.

73. The method of any one of the preceding clauses, wherein the step ofcontacting the cell-free DNA composition occurs in reaction volume thatis less than 40 μL.

74. The method of any one of the preceding clauses, wherein the step ofcontacting the cell-free DNA composition occurs in reaction volume thatis less than 30 μL.

75. The method of any one of the preceding clauses, wherein the step ofcontacting the cell-free DNA composition occurs in reaction volume thatis less than 20 μL.

76. The method of any one of the preceding clauses, wherein the fetalDNA is about 30% of total DNA in the nucleic acid sample.

77. The method of any one of the preceding clauses, wherein the fetalDNA is no greater than 30% of total DNA in the nucleic acid sample.

78. The method of any one of the preceding clauses, wherein the fetalDNA is more than 30% of total DNA in the nucleic acid sample.

79. A kit useful in the detection of fetal copy number variationcomprising:

a. a capture device having a plurality of nucleic acid fragmentscorresponding at least one chromosomal target region attached thereto;b. a plurality of molecular probes capable of hybridizing to a mixednucleic acid population that includes a major subpopulation and a minorsubpopulation, wherein the major and minor subpopulations each include atarget sequence located in a first chromosomal region and containing apolymorphic site, wherein the polymorphic site can include combinationsof a first nucleotide variant and a second nucleotide variant; and c.instructions for genotyping and detecting the polymorphic site.

80. The kit of clause 77, wherein the capture device is a microarray.

81. The kit of clause 77 or 78, wherein the chromosomal target region ison one or more of chromosomes 1, 5, 13, 18, 21, X, and Y.

82. The kit of any one of clauses 77 to 79, wherein molecular probes aredesigned to genotype a single nucleotide polymorphism on one or more ofchromosomes 1, 5, 13, 18, 21, X, and Y.

83. A method for detecting a copy number in a fetus, comprising:obtaining a biological sample from a subject who is a pregnant female,the biological sample including nucleic acid of both maternal and fetalorigin containing a target nucleic acid sequence located on a firstchromosome, the target nucleic acid sequence containing a polymorphicsite for a single nucleotide polymorphism (SNP); generating a populationof nucleic acid fragments containing or derived from the target nucleicacid sequence; conducting a first assay comprising (a) contacting thepopulation of nucleic acid fragments with an oligonucleotide arraycontaining a first oligonucleotide probe configured to hybridize to thetarget nucleic acid sequence containing the polymorphic site of the SNP;and (b) detecting, using a detector, first signals indicatinghybridization of the oligonucleotide probe to one or more nucleic acidfragments of the population containing a first allelic variant (“Aallele”) of the SNP and second signals indicating hybridization of theoligonucleotide probe to one or more nucleic acid fragments of thepopulation containing a second allelic variant (“B allele”) of the SNP;and determining, using the first signals and the second signals, any oneor more of the following: (i) the copy number of the first chromosome inthe fetus; (ii) a fetal genotype for the SNP; (iii) a maternal genotypefor the SNP; and (iv) a fetal fraction of the sample.

84. The method of clause 83, further comprising calculating the observedB-allele frequency (BAF) for the allelic variants of the SNP present inthe sample.

85. The method of clause 84, further including calculating the fetalfraction of the sample using the BAF.

86. The method of clause 84, wherein the polymorphic site of the SNP canbe homozygous for the A allele (“AA”), homozygous for the B allele(“BB”) or heterozygous (“AB”).

87. The method of any of clauses 83-86, wherein the detector has a firstand a second detection channel, and the genotyping further includesdetecting the first signals in the first channel and the second signalsin the second channel.

88. The method of clause 87, wherein the first signals in the firstchannel indicate the amount of A allele present in the nucleic acidpopulation and the second signals indicate the amount of B allelepresent at nucleic acid population.

89. The method of clause 88, wherein determining the copy number of thefirst chromosome in the fetus includes determining a ratio of a firstvalue to a second value.

90. The method of clause 86, further including determining a firstmaternal SNP genotype.

91. The method of clause 83, wherein the nucleic acid sample includesmaternal blood, plasma or serum and the nucleic acid of both maternaland fetal origin includes cell-free DNA (cfDNA).

92. The method of clause 83, wherein the fetal DNA is no greater than20% of total DNA in the nucleic acid sample.

93. The method of clause 83, wherein the fetal DNA is no greater than15% of total DNA in the nucleic acid sample.

94. The method of clause 83, wherein the fetal DNA is no greater than10% of total DNA in the nucleic acid sample.

95. The method of clause 83, wherein the fetal DNA is no greater than 5%of total DNA in the nucleic acid sample.

96. The method of clause 83, wherein the fetal DNA is about 30% of totalDNA in the nucleic acid sample.

97. The method of clause 83, wherein the fetal DNA is no greater than30% of total DNA in the nucleic acid sample.

98. The method of clause 83, wherein the fetal DNA is more than 30% oftotal DNA in the nucleic acid sample.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a diagrammatic view of a method for analyzing a mixed nucleicacid sample in accordance with the present disclosure, showing that themixed nucleic acid sample is split into an A/T channel and a C/G channeland then recombined several steps later for hybridization and stainingsteps.

FIG. 2 is a diagrammatic view of a molecular inversion probe (MIP) usedin a method in accordance with the present disclosure.

FIG. 3 is diagrammatic views of a MIP process showing from left to righta MIP binding to a nucleic acid over a SNP position, the SNP positionbeing gap-filled and ligated to form a circularized MIP, treating thenucleic acid sample with an exonuclease, cleaving the circularized MIP,amplifying a portion of the cleaved MIP, and digesting the amplifiedproduct.

FIG. 4 is a diagrammatic view of hybridizing and staining the amplifiedproduct shown in FIG. 3 using an oligonucleotide array, showing theamplified product hybridized to a probe on the oligonucleotide array,and further showing detecting the hybridized product with either a firstor second dye.

DETAILED DESCRIPTION

The present disclosure has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

Throughout this disclosure, various aspects of this disclosure can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present disclosure may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

Definitions

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

All references cited herein are incorporated herein in their entiretiesfor all their purposes. To the extent any reference includes adefinition or uses a claim term in a manner inconsistent with thedefinitions and disclosure set forth herein, the definitions anddisclosure of this application will control.

As used herein, “allele” refers to one specific form of a nucleic acidsequence (such as a gene) within a cell, an individual or within apopulation, the specific form differing from other forms of the samegene in the nucleic acid sequence of at least one, and frequently morethan one, variant sites within the sequence of the gene. The sequencesat these variant sites that differ between different alleles are termed“variances”, “polymorphisms”, or “mutations”. The variants in thesequence can occur as a result of SNPs, combinations of SNPs, haplotypemethylation patterns, insertions, deletions, and the like. An allele maycomprise the variant form of a single nucleotide, a variant form of acontiguous sequence of nucleotides from a region of interest on achromosome, or a variant form of multiple single nucleotides (notnecessarily all contiguous) from a chromosomal region of interest. Ateach autosomal specific chromosomal location or “locus” an individualpossesses two alleles, one inherited from one parent and one from theother parent, for example one from the mother and one from the father.An individual is “heterozygous” at a locus if it has two differentalleles at that locus. An individual is “homozygous” at a locus if ithas two identical alleles at that locus.

As used herein, “an array” or “a microarray” comprises a support,preferably solid, with nucleic acid probes attached to the support.Preferred arrays typically comprise a plurality of different nucleicacid probes that are coupled to a surface of a substrate in different,known locations. These arrays, also described as “microarrays” orcolloquially “chips” have been generally described in the art, forexample, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195,5,800,992, 6,040,193, 5,424,186 and Fodor et al., Science, 251:767-777(1991). Each of which is incorporated by reference in its entirety forall purposes. The probes can be of any size or sequence, and can includesynthetic nucleic acids, as well as analogs or derivatives ormodifications thereof, as long as the resulting array is capable ofhybridizing under any suitable conditions with a nucleic acid samplewith sufficient specificity as to discriminate between different targetnucleic acid sequences of the sample. In some embodiments, the probes ofthe array are at least 5, 10 or 20 nucleotides long. In someembodiments, the probes are no longer than 25, 30, 50, 75, 100, 150, 200or 500 nucleotides long. For example, the probes can be between 10 and100 nucleotides in length.

Arrays may generally be produced using a variety of techniques, such asmechanical synthesis methods or light directed synthesis methods thatincorporate a combination of photolithographic methods and solid phasesynthesis methods. Techniques for the synthesis of these arrays usingmechanical synthesis methods are described in, e.g., U.S. Pat. Nos.5,384,261, and 6,040,193, which are incorporated herein by reference intheir entirety for all purposes. Although a planar array surface ispreferred, the array may be fabricated on a surface of virtually anyshape or even a multiplicity of surfaces. Arrays may be nucleic acids onthree-dimensional matrices, beads, gels, polymeric surfaces, fibers suchas optical fibers, glass or any other appropriate substrate. (See U.S.Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992,which are hereby incorporated by reference in their entirety for allpurposes.)

In some embodiments, arrays useful in connection with the methods andsystems described herein include commercially available from ThermoFisher Scientific (formerly Affymetrix) under the brand name GeneChip®and are directed to a variety of purposes, including genotyping and geneexpression monitoring for a variety of eukaryotic and prokaryoticspecies. Methods for preparing a sample for hybridization to an arrayand conditions for hybridization are disclosed in the manuals providedwith the arrays, for example, those provided by the manufacturer inconnection with products, such as the OncoScan® FFPE Assay Kit, andrelated products.

As used herein, “cell-free nucleic acid” means nucleic acid molecules ofpresent in the body of an organism but that are not contained within anyintact cells. The cell-free nucleic acid can include DNA (“cell-freeDNA”) or RNA (“cell-free RNA”) or derivatives or analogs thereof. Thecell-free nucleic acid can be obtained from blood, plasma, saliva, orurine. The cell-free DNA or RNA can include circulating cell-free DNA orRNA, i.e., cell-free DNA or RNA found in the plasma fraction of blood.

It will be appreciated that numerous methods and kits are known to oneof skill in the art for the purpose of obtaining cell-free DNA from asample, such as human blood plasma, serum, urine, stool or saliva.

As used herein, “genome” designates or denotes the complete, single-copyset of genetic instructions for an organism as coded into the DNA of theorganism. A genome may be multi-chromosomal such that the DNA iscellularly distributed among a plurality of individual chromosomes. Forexample, in humans there are 22 pairs of chromosomes plus a genderassociated XX or XY pair.

As used herein, “genotyping” refers to the determination of the nucleicacid sequence information from a nucleic acid sample at one or morenucleotide positions. The nucleic acid sample may contain or be derivedfrom any suitable source, including the genome or the transcriptome. Insome embodiments, genotyping may comprise the determination of whichallele or alleles an individual carries at one or more polymorphicsites. For example, genotyping may include or the determination of whichallele or alleles an individual carries for one or more SNPs within aset of polymorphic sites. For example, a particular nucleotide in agenome may be an A in some individuals and a C in other individuals.Those individuals who have an A at the position have the A allele andthose who have a C have the B allele. In a diploid organism theindividual will have two copies of the sequence containing thepolymorphic position so the individual may have an A allele and a Ballele or alternatively two copies of the A allele or two copies of theB allele. Those individuals who have two copies of the B allele arehomozygous for the B allele, those individuals who have two copies ofthe A allele are homozygous for the B allele, and those individuals whohave one copy of each allele are heterozygous. The array may be designedto distinguish between each of these three possible outcomes. Apolymorphic location may have two or more possible alleles and the arraymay be designed to distinguish between all possible combinations. Insome embodiments, one or more polynucleotides (or a portion or portionsof the polynucleotide, its amplification products, or complementsthereof) that contain a sequence of interest (e.g., one or more SNP ormutation) can be processed by other techniques such as sequencing.Therefore, in some embodiments, the polynucleotides can be sequenced forgenotyping or determining the presence or absence of the polymorphism ormutation. The sequencing can be done via various methods available inthe art, e.g., Sanger sequencing method that can be performed by, e.g.,SeqStudio® Genetic Analyzer from Applied Biosystems) or Next GenerationSequencing (NGS) method, e.g., Ion Torrent NGS from Thermo Fisher orIllumina NGS. In some embodiments, genotyping includes detecting asingle nucleotide mutation that arises spontaneously in the genome,amongst a background of wild-type nucleic acid. In some embodiments,genotyping includes determining fetal blood type from a sample ofmaternal blood.

The term “chromosome” refers to the heredity-bearing gene carrier of aliving cell which is derived from chromatin and which comprises DNA andprotein components (especially histones). The conventionalinternationally recognized individual human genome chromosome numberingsystem is employed herein. The size of an individual chromosome can varyfrom one type to another with a given multi-chromosomal genome and fromone genome to another. In the case of the human genome, the entire DNAmass of a given chromosome is usually greater than 100,000,000 bp. Forexample, the size of the entire human genome is about 3×10⁹ bp. Thelargest chromosome, chromosome no. 1, contains about 2.4×10⁸ bp whilethe smallest chromosome, chromosome no. 22, contains about 5.3×10⁷ bp.In some embodiments, chromosomes of interest in connection with themethods and systems of the present disclosure include those chromosomesthat are associated with a chromosomal abnormality, such as chromosomes13, 18, 21, X, and Y. It will be further appreciated that otherchromosomes not associated with a particular chromosomal abnormality,such as aneuploidy, can be of interest in connection with the methodsand systems of the present disclosure as reference chromosomes. It willbe appreciated that a reference chromosome can be any of the chromosomesin a genome that are not associated with a particular chromosomalabnormality, such as aneuploidy, such as chromosomes 1 and 5.

As used herein, “chromosomal region” means a portion of a chromosome.The actual physical size or extent of any individual chromosomal regioncan vary greatly. The term “region” is not necessarily definitive of aparticular one or more genes because a region need not take intospecific account the particular coding segments (exons) of an individualgene. In some embodiments, a chromosomal region will contain at leastone polymorphic site.

As used herein, “chromosomal abnormalities” or “chromosomal abnormality”can include any genetic abnormality including but not limited toaneuploidy, such as trisomy 21 (a.k.a. Down syndrome); trisomy 18(a.k.a. Edwards syndrome); trisomy 13 (a.k.a. Patau syndrome); XXY(a.k.a. Klinefelter's syndrome); monosomy 18; X (a.k.a. Turnersyndrome); XYY (a.k.a. Jacobs Syndrome), or XXX (a.k.a. Trisomy X);trisomy associated with an increased chance of miscarriage (e.g.,Trisomy 15, 16, or 22); and the like, as well as other geneticvariations, such as mutations, insertions, additions, deletions,translocation, point mutation, trinucleotide repeat disorders and/orSNPs. While the present disclosure describes certain examples andembodiments related to the detection of chromosomal abnormalities in afetus, it will be appreciated that the methods and system describedherein can be used to detect chromosomal abnormalities in other diseasestates, such as cancer.

As used herein, “maternal sample” can be any sample taken from apregnant mammal which comprises both fetal and maternal cell-free DNA.Preferably, maternal samples for use in connection with the presentdisclosure are obtained through relatively non-invasive means, e.g.,phlebotomy, saliva or urine collection, or other standard techniques forextracting peripheral samples from a subject.

As used herein “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes ribonucleoside triphosphatesATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates (dNTPs) such asdATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Suchderivatives include, for example, [αS]dATP, 7-deaza-dGTP and7-deaza-dATP, and nucleotide derivatives that confer nuclease resistanceon the nucleic acid molecule containing them. The term nucleotide asused herein also refers to dideoxyribonucleoside triphosphates (ddNTPs)and their derivatives. Illustrated examples of dideoxyribonucleosidetriphosphates include, but are not limited to, ddATP, ddCTP, ddGTP,ddITP, and ddTTP.

As used herein, “polymorphism” refers to the occurrence of two or moregenetically determined alternative sequences in a population. Thealternative sequences can include alleles (e.g., naturally occurringvariants) or spontaneously arising mutations that only occur in one orfew individual organisms. A “polymorphic site” can refer to the nucleicacid position(s) at which a difference in nucleic acid sequence occurs.A polymorphism may comprise one or more base changes, an insertion, arepeat, or a deletion. A polymorphic locus may be as small as one basepair. Polymorphic sites include restriction fragment lengthpolymorphisms, variable number of tandem repeats (VNTR's), hypervariableregions, minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, and insertionelements. The first identified variant or allelic form is arbitrarilydesignated as the reference form and other variant or allelic forms aredesignated as alternative or variant or mutant alleles. The variant orallelic form occurring most frequently in a selected nucleic acidpopulation is sometimes referred to as the wildtype form. In someembodiments, the wildtype form can be referred to as a “majorsubpopulation” and the mutant can be referred to as ta “minorsubpopulation”. In some embodiments, the more frequently occurringallele can be referred to as a “major subpopulation” and the rarer orless frequently occurring allele can be referred to as ta “minorsubpopulation”. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms. A polymorphism between two nucleic acidscan occur naturally, or be caused by exposure to or contact withchemicals, enzymes, or other agents, or exposure to agents that causedamage to nucleic acids, for example, ultraviolet radiation, mutagens orcarcinogens. SNPs are positions at which two alternative bases occur atappreciable frequency (>1%) in the human population, and are the mostcommon type of human genetic variation.

As used herein, “sample obtained from an organism” includes but is notlimited to any number of tissues or fluids, such as blood, urine, serum,plasma, lymph, saliva, stool, and vaginal secretions, of virtually anyorganism. In some embodiments, a sample obtained from an organism can bea mammalian sample. And in some embodiments, a sample obtained from anorganism can be a human sample. In some embodiments, a sample obtainedfrom an organism can be a maternal sample.

Genotyping

In some embodiments, the methods described in the present disclosureinclude a step of genotyping. The genotyping can include determining thesequence of at least one nucleotide within a target nucleic acidsequence. In some embodiments, the step of genotyping involves analyzinga mixed nucleic acid population that includes a major subpopulation anda minor subpopulation, wherein the major and minor subpopulations eachinclude a target sequence located in a first chromosomal region andcontaining a polymorphic site. In some embodiments, the methodsdescribed herein are used to genotype the major subpopulation. In someembodiments, the methods described herein are used to genotype the minorsubpopulation. In some embodiments, the methods described herein areused to genotype both the major subpopulation and the minorsubpopulation.

It will be appreciated that genotyping can be carried out in any manneruseful for the identification of polymorphic sites in a target sequenceof a nucleic acid sample. In some embodiments, methods of genotypinguseful in connection with the present disclosure include those methodsuseful for SNP detection. Platforms for SNP detection are well known inthe art. Suitable methods for genotyping include variations of singlenucleotide extension, use of allele-specific probes, ligation-basedallelic discrimination, and the like.

In the context of array based assays, a variety of genotyping methodsare available. In some embodiments, the array surface is divided intofeatures, each feature containing multiple sites that include copies ofsubstantially identical oligonucleotides configured to bind to aparticular target nucleic acid sequence. Hybridization of nucleic acidmolecules to different locations on the array can be detected andquantified. One suitable method is to use any array containingallele-specific probes that selectively bind only to certain alleles andnot others. In other embodiments, the array contains probes that bindnon-selectively to all of the different forms of an allele, but then isextended or otherwise modified in an allele-specific manner to generatean allele-specific product. For example, the probe of the array can beelongated via template-dependent nucleotide polymerization.Alternatively, the probe can be elongated via sequence-dependentligation of a tag oligonucleotide, which may contain a signal-generatingmoiety. In still, allele-specific products (e.g., allele-specificnucleotide extension products or ligation products) can be generatedoff-array, and then hybridized to an array containing probes thatdiscriminate between the various extension products. Signals emittedfrom the array indicating hybridization of nucleic acid molecules tospecific array probes can be detected and quantified. Examples ofgenotyping array products include the Affymetrix Axiom® arrays and theAffymetrix OncoScan arrays (Thermo Fisher Scientific) as well asIllumina's BeadChip® and Infinium® arrays, Suitable array-basedgenotyping methods are described, for example, in Hoffman et al,Genomics 98(2):79-89 (2011) and Shen et al., Mutation Research 573:70-82(2005), both of which are incorporated herein in their entireties.

One method useful for genotyping variations in nucleic acid sequence(including through use of microarrays) is the molecular inversion probe(MIP) assay. See for example, U.S. Pat. No. 6,858,412, incorporatedherein by reference in its entirety pertaining to the implementation ofthe MIP assay generally.

In general MIP probes include at least a 5′-target sequence, a 3′-targetsequence, a 5′-primer site and/or a 3′-primer site, a tag sequence, andone or more cleavage sites. In one exemplary embodiment, MIP probesuseful in connection with the present disclosure can be represented asshown in FIG. 2. The MIP probe of FIG. 2 includes genomic homology 1 andgenomic homology 2 that correspond to target a sequence on a chromosomalregion that is a known SNP locus. Genomic homology 1 and genomichomology 2 are designed to have a one nucleotide gap in the probe afterthe probe has been hybridized to a nucleic acid fragment (e.g. acell-free DNA fragment). In addition to genomic homology 1 and genomichomology 2 the MIP probes useful in connection with some embodiments ofthe disclosure include a first primer binding site and a second primerbinding site, a tag sequence and two cleavage sites.

It will be appreciated that pools of MIP probes can be applied to themethods and systems described herein for multiplex detection of SNPs inthe mixed nucleic acid sample. For example, in some embodiments, a poolof MIP probes can be pulled from the commercially available MIP probessets used in connection with the OncoScan® product available from ThermoFisher Scientific. For example, in some embodiments, a pool of about48,000 MIP probes corresponding to SNP loci in chromosomes 13, 18, 21,X, and Y can be pulled from the OncoScan® product. In addition, it willbe appreciated that additional pools of MIP probes, such as thosecorresponding to SNP loci on chromosomes 1 and 5 can be pulled from theOncoScan® product for use as reference probes. In some embodiments, atleast 50%, at least 60%, or at least 70% of the MIPS in the pool of MIPprobes bind to DNA fragments from chromosomes 1, 5, 13, 18, 21, X, andY.

In some embodiments, the pool of MIP probes comprises at least 1,000MIPS, at least 5,000, at least 10,000, or at least 20,000 MIPS. In someembodiments, the pool of MIP probes comprises less than 200,000, lessthan 100,000, or less than 80,000 MIPS.

An exemplary MIP assay process useful in connection with the presentdisclosure is shown in FIG. 3. Briefly, the MIP probe can be hybridizedto a target sequence located in a first chromosomal region containing apolymorphic site in an annealing step. The annealing step can be carriedout according to any method commonly known in the art, especiallyaccording to manufacturer instructions for a commercially available MIPprobe set. The step of annealing provides a plurality of linearmolecular inversion probe-DNA fragment complexes, such that the genomichomology 1 and genomic homology 2 sequences hybridize to the chromosomalregion containing a polymorphic site with a one nucleotide gap betweenthe ends of the hybridized probe.

In some embodiments, the total amount of DNA fragments or mixed nucleicacid population is less than 50 ng, less than 40 ng, less than 30 ng,less than 20 ng, less than 15 ng, or less than 10 ng. In someembodiments, the ratio of the total number of MIPS to the total numberof DNA fragment copies is at least about 15,000:1 or at least about30,000:1. In some embodiments, the ratio of the total number of MIPS tothe total number of DNA fragment copies is less than 100,000:1 or lessthan 60,000:1. In some embodiments, the ratio of the total number ofMIPS to the total number of DNA fragment copies is about 40,000:1.

In some embodiments, the annealing step is performed in a reactionvolume that is less than 50 μL, less than 40 μL, less than 30 μL, lessthan 20 μL, or less than 15 μL. In some embodiments, the reaction volumeis at least 5 μL or at least 10 μL.

In some embodiments, the mixed nucleic acid population contains or isderived from cell-free DNA present in blood, srum and/or plasma of theorganism at a concentration of no greater than 5 ng/mL and no less than0.1 ng/mL. In some embodiments, the mixed nucleic acid populationcontains or is derived from cell-free DNA present in blood, srum and/orplasma of the organism at concentration of less than 5 ng/mL, less than4 ng/mL, less than 3 ng/mL, less than 2 ng/mL, less than 1 ng/mL, lessthan 0.5 ng/mL, or less than 0.3 ng/mL. In some embodiments, the mixednucleic acid population contains or is derived from cell-free DNApresent in blood, srum and/or plasma of the organism at concentration ofgreater than 0.1 ng/mL, greater than 0.2 ng/mL, greater than 0.3 ng/mL,greater than 0.5 ng/mL, greater than 1 ng/mL, greater than 2 ng/mL, orgreater than 3 ng/mL.

After the annealing step is completed, the annealing mixture may or maynot be separated into a first channel and a second channel, depending onthe particular genotyping application. In some embodiments, theannealing mixture can be separated into a first channel and a secondchannel (as shown in FIG. 1). In such an embodiment, the annealingmixture is split into a first channel composition and a second channelcomposition that can be carried forward through genotyping process. Insome embodiments, the annealing mixture is not split into a firstchannel and a second channel, but rather carried on as a singlereaction.

In some embodiments, the annealing mixture can be subjected to aligation step, also referred to as a “gap-fill” step to incorporatenucleotides in the gap between genomic homology 1 and genomic homology 2of the linear MIP, as shown in FIG. 3. For the gap fill reaction, anyknown method in the art will suffice. For example, a mix ofdeoxynucleotides (dATP, dCTP, dGTP, dTTP, dUTP) can be added to areaction mix, as well as a polymerase, ligase and other reactioncomponents and incubating at about 60° C. for about 10 minutes, followedby incubation at 37° C. for about 1 minute. Following annealing andligation, the MIP may become circularized. In some embodiments, thenucleotides added to the first and second channel may be the same ordifferent.

In some embodiments, where it is advantageous to add different sets ofdeoxynucleotides to the gap-fill reaction, the deoxynucleotides added toone of the channels can be dATP and dTTP, while the deoxynucleotidesadded to one of the other channels can be dCTP and dGTP. It will beappreciated that the different deoxynucleotide mixtures can be added toeither channel. In this way, each channel can selectively detectdifferent SNP alleles in a first circularized probe composition and asecond circularized probe composition. In some embodiments, a channelmay be substantially free of dGTP, dCTP, or a mixture thereof. In someembodiments, a channel may be substantially free of dATP, dTTP, or amixture thereof.

It will be appreciated that the ligase used in the gap-fill step is notparticularly preferred, and can be any ligase known in the art, andaccording to any standard protocol known in the art. Many ligases areknown and are suitable for use in the connection with the presentdisclosure for the gap-fill reaction. See for example, Lehman, Science,186: 790-797 (1974); Engler et al, DNA Ligases, pages 3-30 in Boyer,editor, The Enzymes, Vol. 15B (Academic Press, New York, 1982); and thelike. Optional ligases for use in connection with the MIP gap-fillreaction include, but are not limited to, T4 DNA ligase, T7 DNA ligase,E. coli DNA ligase, Taq ligase, Pfu ligase, and Tth ligase. Protocolsfor use of such ligases are well known (See for example, Barany, PCRMethods and Applications, 1: 5-16 (1991); Marsh et al, Strategies, 5:73-76 (1992); and the like). In some embodiment, the ligase can be athermostable or (thermophilic) ligase, such as pfu ligase, Tth ligase,Taq ligase and Ampligase™ DNA ligase (Epicentre Technologies, Madison,Wis.).

In some embodiments, the respective circularized probe compositions,when there are more than one, can be subjected to an exonucleasedigestion step, as shown in FIG. 3. The purpose of the exonucleasedigestion step is to digest/remove any remaining nucleic acid fragmentsfrom the nucleic acid sample obtained from an organism, and todigest/remove any remaining uncircularized MIPs. It will be appreciatedthat such an optional digestion step can improve later PCR amplificationby removing nucleic acid fragments that may interfere with the PCRreaction, or may form chimeric products that interfere with furtherprocessing of the sample later in the process. Suitable 3′-exonucleasesinclude, but are not limited to, exo I, exo III, exo VII, exo V, andpolymerases, as many polymerases have excellent exonuclease activity,etc.

After optional removal of uncircularized MIPS and DNA fragments, thecircularized probes, in some embodiments, first circularized probes andsecond circularized probes can be cleaved to form to form a firstlinearized probe composition and a second linearized probe composition.It will be appreciated that the cleaving can be accomplished accordingto any method known in the art suitable for use in connection with thepresent teachings. In some embodiments, one or more circulized probes,e.g., the first and/or second circulizaed probes are single-stranded. Insome embodiments, the circulaized probe(s) is/are double-stranded. Insome embodiments, the circualized probes are cleaved to form linearizedprobes. In some embodiments, there are one or more enzymes to be used tolinearize the probes. In some embodiments, an enzyme that is capable ofcleaving a single-stranded nucleic acid can be used to linearize theprobes. In some embodiments, such an enzyme cleaving a single-strandednucleic acid is uracil-N-glycosylase. In some other embodiments, one ormore restriction enzymes can be used to linearize the probes. In someembodiments, the step of cleaving can be catalyzed by adding an ezymesuch as uracil-N-glycosylase or a restriction enzyme to the linearizedprobe composition, and in some embodiments, the first and secondlinearized probe composition, cleaving the circular probes to form afirst linearized probe composition and a second linearized probecomposition. Suitable restriction enzymes include, but are not limitedto AatII, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI,AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AseI,AsiSI, AvaI, AvaII, AvrII, BaeGI, BacI, BamHI, BanI, BanII, BbsI, BbvCI,BbvI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BfuAI, BfuCI, BglI, BglII,BlpI, BmgBI, BmrI, BmtI, BpmI, Bpul0I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI,BsaJI, BsaWI, BsaXI, BscRI, BscYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI,BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI,BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssKI, BssSI,BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I,BtgI, BtgZI, BtsCI, BtsI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI,DdcI, DpnI, DpnII, DraI, DraIII, DrdI, EacI, EagI, EarI, EciI, Eco53kI,EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI, FauI, Fnu4HI, FokI, FseI,FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinPlI, HpaI,HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I, HpyAV, HpyCH4III,HpyCH4IV, HpyCH4V, KasI, KpnI, MboI, MboII, MfeI, MluI, MlyI, MmeI,MnII, MscI, MseI, MsII, MspAlI, MspI, MwoI, NaeI, NarI, Nb.BbvCI,Nb.BsmI, Nb.BsrDI, Nb.BtsI, NciI, Ncof, NdeI, NgoMIV, NheI, NlaIII,NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI,Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaeR7I, PciI, PflFI, PflMI, PhoI,PleI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI,PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI,ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, SpeI,SphI, SspI, StuI, StyD4I, StyI, SwaI, T, TagαI, TfiI, TliI, TseI,Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI,and ZraI. It will be appreciated that the MIP probe can be designed tocontain one or more, and in some embodiments two, restriction sites. Inthe case where MIPs are designed with two restriction sites, one ofskill in the art will understand how to design the MIPs such that therestriction enzymes will act selectively on each cleavage site of theMIP.

As mentioned above, the MIP probe can be designed with one or two primersites. As used herein, a “universal priming site” is a site to which auniversal primer will hybridize. In general, “universal” refers to theuse of a single primer or set of primers for a plurality ofamplification reactions. For example, in the detection or genotyping ofa 100 different target sequences, all the MIPs may share the identicaluniversal priming sequences, allowing for the multiplex amplification ofthe 100 different probes using a single set of primers. This allows forease of synthesis (e.g. only one set of primers is made), resulting inreduced costs, as well as advantages in the kinetics of hybridization.Most importantly, the use of such primers greatly simplifiesmultiplexing in that only two primers are needed to amplify a pluralityof probes. In general, the universal priming sequences/primers eachrange from about 12 to about 40 base pairs in length. Suitable universalpriming sequences are known to one of skill in the art, and specificallyinclude those exemplified herein. In some embodiments, the MIP is alsodesigned with a tag sequence, or a barcode sequence, that will allow forspecific detection of two channel probes using a two-color system. Insuch an example, the universal primer sequence at one end of thelinearized probes, either the 5′- or 3′-end, depending on theapplication and the detection platform, will contain a specific sequenceto recognize a particular colored label. Thus it can be advantageous todesign a MIP to have a restriction site between two universal 3′- and5′-ends of universal primers.

Once the circularized probes are cleaved to form linearized probes, theprobes can be subjected to an amplifying step of the first linearizedprobe composition in the presence of a first tailed primer to form afirst amplified product composition, and amplifying the secondlinearized probe composition in the presence of a second tailed primerto form a second amplified product composition, wherein the first tailedprimer has a tail sequence that is different from the second tailedprimer. The amplification step can be carried out by any method known inthe art. The PCR reaction can be carried out in the presence of apolymerase useful in connection with the present disclosure, such as USDTaq. In some embodiments, the amplification step is carried out in thepresence of a hot-start polymerase comprising the polymerase and apolymerase inhibitor. In some embodiments, the polymerase inhibitor isdisassociated from the polymerase when the temperature is at least 40°C. In some embodiments, the amplification step is carried out in thepresence of Titanium Taq polymerase. In some embodiments, theamplification step is carried out in the presence of Platinum SuperFiDNA Polymerase.

The present disclosure also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, and each of which is incorporated herein by reference intheir entireties for all purposes. The sample may be amplified on thearray. See, for example, U.S. Pat. No. 6,300,070 and U.S. patentapplication Ser. No. 09/513,300, which are incorporated herein byreference.

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleic acidsequences (U.S. Pat. No. 6,410,276), consensus sequence primedpolymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975),arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos.5,413,909, 5,861,245) and nucleic acid based sequence amplification(NASBA). (See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, eachof which is incorporated herein by reference). Other amplificationmethods that may be used include: Qbeta Replicase, described in PCTPatent Application No. PCT/US87/00880, isothermal amplification methodssuch as SDA, described in Walker et al. 1992, Nucleic Acids Res.20(7):1691-6, 1992, and rolling circle amplification, described in U.S.Pat. No. 5,648,245. Other amplification methods that may be used aredescribed in U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317, U.S. Pat. Nos. 8,673,560 and 8,728,728 and US Pub.No. 20030143599, each of which is incorporated herein by reference. Insome embodiments, DNA is amplified by multiplex locus-specific PCR. Forexample, the DNA can be amplified using Thermo Fisher's AmpliSeq®products. In one embodiment, the DNA is amplified using adaptor-ligationand single primer PCR. Other available methods of amplification, such asbalanced PCR (Makrigiorgos, et al. (2002), Nat Biotechnol, Vol. 20, pp.936-9), may also be used.

After the amplification step is complete, the first amplified productcomposition and the second amplified product composition, in embodimentswhere the nucleic acid sample was split into two channels for separateallele detection, can be combined to form an amplified product mixturecomprising first amplified products and second amplified products. Thefirst and second amplified products are then ready for hybridizing andlabelling. In some embodiments, the amplified product compositions thatundergo hybridization and labeling steps can be analyzed via array-baseddetection. Alternatively, the amplified product compositions can beprocessed by other techniques such as conventional or massively parallelsequencing. Thus, in some embodiments, the amplified productcompositions, which can be optionally cleaved as described below,proceed to sequencing-based detection. The sequencing can be done viavarious methods available in the field, e.g., methods involvingincorporating one or more chain-terminating nucleotides, e.g., SangerSequencing method that can be performed by, e.g., SeqStudio® GeneticAnalyzer from Applied Biosystems. In other embodiments, the sequencingcan include performing a Next Generation Sequencing (NGS) method, e.g.,primer extension followed by semiconductor-based detection (e.g., IonTorrent™ systems from Thermo Fisher Scientific) or via fluorescentdetection (e.g., Illumina systems).

In some embodiments, after the amplification is complete, the amplifiedproduct compositions can be cleaved with one or more enzymes. In someembodiments, the amplified product compositions, e.g., the first and/orsecond amplified product compositions have a restriction enzymerecognition site. In some embodiments, the step of cleaving can becatalyzed by adding a restriction enzyme to the amplified productcompositions, and in some embodiments, the first and second amplifiedproduct compositions. Suitable restriction enzymes include, but are notlimited to AatII, Acc65I, AccI, AclI, AclI, AcuI, AfeI, AflII, AflIII,AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI,AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI,BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BfuAI, BfuCI, BglI,BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpul0I, BpuEI, BsaAI, BsaBI,BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BscRI, BscYI, BsgI, BsiEI, BsiHKAI,BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI,BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII,BssKI, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI,BstZ17I, Bsu36I, BtgI, BtgZI, BtsCI, BtsI, Cac8I, ClaI, CspCI, CviAII,CviKI-1, CviQI, DdcI, DpnI, DpnII, DraI, DraIII, DrdI, EacI, EagI, EarI,EciL, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, FatI, FauI,Fnu4HI, FokI, FseI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII,HinfI, HinPlI, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hpy99I,HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, KasI, KpnI, MboI, MboII, MfeI,MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspAlI, MspI, MwoI, NaeI,Narf, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, NciI, NcoI, NdeI, NgoMIV,NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI,Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaeR7I, PciI, PflFI,PflMI, PhoI, PleI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI,PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I,SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI,SnaBI, SpeI, SphI, SspI, StuI, StyD4I, StyI, SwaI, T, TaqαI, TfiI, TliI,TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI,XmnI, and ZraI. In some embodiments, the restriction enzyme used tocleave the first and second amplified product compositions are identicalor different. In some embodiments where the same restriction enzyme isused to cleave the first and second amplified product compositions, therestriction enzyme HaeIII is used to cleave its specific site present inthe first and second amplified products. It will be appreciated that theamplified product composition which contains amplified MIP probes of thedisclosure can be designed to contain one or more restriction sites. Inthe case where MIPs are designed with two or more restriction sites, oneof skill in the art will understand how to design the MIPs such that therestriction enzymes will act selectively on each cleavage site of theMIP. In some embodiments, the cleavage of the amplified productcompositions occur before or after combining the first and secondamplified product compositions to form an amplified product mixture.

Detecting

The step of hybridizing at least one nucleic acid fragment containing orderived from the nucleic acid population and containing the polymorphicsite to an oligonucleotide probe of an oligonucleotide array can beaccomplished according to any known method in the art, and specificallyin connection with the instructions received with any platform useful inconnection with the present disclosure, such as the Axiom 2.0 reagentkit.

In some embodiments, the step of hybridization further includes a stepof fixing. The fixing can include contacting the oligonucleotide arraywith nucleic acid hybridized thereto with a suitable fixing agent. Insome embodiments, the fixing step occurs after the hybridization step iscompleted. In some embodiments, the fixing step occurs well after thehybridization step, e.g., after the hybridized array is washed andstained with a strain mixture. Therefore, in some embodiments, the arraymay undergo the steps of hybridization, washing, staining and fixing inthis order, along with other steps.

The different primer pair amplified sequences can be differentiatedbased on spectrally distinguishable probes (e.g. 2 different dye-labeledprobes such as Taqman or Locked Nucleic Acid Probes (Universal ProbeLibrary, Roche)). In such approach, all probes are combined into asingle reaction volume and distinguished based on the differences in thecolor emitted by each probe. For example, the probes targeting onepolynucleotide (e.g., a test chromosome, such as chromosome 21) may beconjugated to a dye with a first color and the probes targeting a secondpolynucleotide (e.g., a reference chromosome, such as chromosome 1) inthe reaction may be conjugated to a dye of a second color. The ratio ofthe colors then reflects the ratio between the test and the referencechromosome.

Illustratively, the first and second amplified product compositionscomprise a nucleic acid sequence that, in some embodiments, correspondsto a channel composition. As an example, the amplified productcomposition from the first channel may comprise a first nucleic acidsequence and the amplified product compositions from the second channelmay comprise a second nucleic acid sequence. Illustratively, the firstand second nucleic acid sequences can bind or hybridize different agentsfor measuring the amount of the amplified product. In some embodiments,the amplified product compositions are directly labeled and measured.

The first and second amplified product compositions can be recombinedand detected on a single array or can be kept separate and detected onat least 2 separate arrays. In embodiments where a single array is used,each of the first and second amplified product compositions can belabeled with a different reporter to allow for first and second productcomposition identification on the array. In embodiments where at least 2arrays are used, the first and second amplified product compositions canbe labeled with the same or different reporters. Exemplarysingle-channel systems include the Affymetrix “Gene Chip,” the Illumina“Bead Chip,” Agilent single-channel arrays, the Applied Microarrays“CodeLink” arrays, and the Eppendorf “DualChip & Silverquant.”

Amplified product composition hybridization to the array can be detecteda variety of ways, including the direct or indirect attachment offluorescent moieties, colorimetric moieties, chemiluminescent moieties,and the like. In some embodiments, probe-target hybridization candetected and quantified by detecting fluorophore-, radio-, silver-, orchemiluminescence-labeled agents to determine relative abundance ofnucleic acid sequences in the target. In some embodiments, the amplifiedproduct composition is directly labeled with a fluorophore-, radio-,silver-, or chemiluminescence-label. Many comprehensive reviews ofmethodologies for labeling DNA provide guidance applicable to generatinglabeled oligonucleotide tags of the present invention. Such reviewsinclude Haugland, Handbook of Fluorescent Probes and Research Chemicals,Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak,DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein,editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press,Oxford, 1991); Wetmur, Critical Reviews in Biochemistry and MolecularBiology, 26: 227-259 (1991); Fung et al, U.S. Pat. No. 4,757,141; Hobbs,Jr., et al U.S. Pat. No. 5,151,507; Cruickshank, U.S. Pat. No.5,091,519. In some embodiments, one or more fluorescent dyes can be usedlabels. Some exemplary dyes are described by Menchen et al, U.S. Pat.No. 5,188,934 (4,7-dichlorofluorscein dyes); Begot et al, U.S. Pat. No.5,366,860 (spectrally resolvable rhodamine dyes); Lee et al, U.S. Pat.No. 5,847,162 (4,7-dichlororhodamine dyes); Khanna et al, U.S. Pat. No.4,318,846 (ether-substituted fluorescein dyes); Lee et al, U.S. Pat. No.5,800,996 (energy transfer dyes); Lee et al, U.S. Pat. No. 5,066,580(xanthene dyes): Mathies et al, U.S. Pat. No. 5,688,648 (energy transferdyes); Maceivicz (U.S. Pat. Application No. 2005/0250147); Faham et al.(U.S. Pat. No. 7,208,295); and the like.

Possible methods of detection include direct detection of a reporter. Insome embodiments, a complementary oligonucleotide to an amplifiedproduct composition comprises either afluorescent/luminescent/chromogenic label or can be subsequently bereacted with additional compounds (e.g., immunostaining, aptamers) togenerate a signal. In some embodiments, instead of hapten-labeled probesfor detection, the labeling probes can have fluorophores conjugateddirectly which would eliminate the antibody-mediated signalamplification.

As described herein, an amplified product composition can be generatedfrom PCR by primers flanking the markers. These amplicons can beproduced singly or in multiplexed reactions. In some embodiments,amplified product compositions can be produced as ss-DNA by asymmetricPCR from one primer flanking the polymorphism or as RNA transcribed invitro from promoters linked to the primers. As an example, a fluorescentlabel can be introduced into amplified product compositions directly asdye-bearing nucleotides or bound after amplification usingdye-streptavidin complexes to incorporated biotin containingnucleotides. Illustratively, for amplified product compositions producedby asymmetric PCR, the reporter (e.g. a fluorescent dye) can be linkeddirectly to the 5′ end of the primer. In some embodiments, amplifiedproduct compositions can be labeled at the 3′ end using TdT and abiotinylated dATP. Illustratively, this could be done for each of theseparate gap fill reactions. In some embodiments, the 3′ labeling usingTdT and a biotinylated ATP leads to a one color, two chip read out.

The amplified product composition is hybridized to the array prior to orduring labeling directly or indirectly with a detection agent. After orduring the step of hybridization, a first agent that binds the firstnucleic acid sequence of the amplified product compositions can beintroduced. The first agent can be configured to bind to the firstnucleic acid sequence present in the amplified products from the firstchannel. In some embodiments, the first agent comprises a complementarysequence to a portion of the first target sequence (e.g. the firstnucleic acid sequence).

Illustratively, the first and second amplified product compositionscomprise a nucleic acid sequence that, in some embodiments, correspondsto a channel composition. As an example, the amplified productcomposition from the first channel may comprise a first nucleic acidsequence and the amplified product compositions from the second channelmay comprise a second nucleic acid sequence.

After or during the step of hybridization, a first agent that binds thefirst nucleic acid sequence of the amplified product compositions can beintroduced. The first agent can be configured to bind to the firstnucleic acid sequence present in the amplified products from the firstchannel. In some embodiments, the first agent comprises a complementarysequence to a portion of the first target sequence (e.g. the firstnucleic acid sequence).

In some embodiments, the first agent comprises the first complementarysequence and a first recognition element conjugated to the firstcomplementary sequence. Illustrative examples of first recognitionelements include fluorophores, biotin, peptide tags, combinationsthereof, or any known acceptable recognition element known in the art.In some examples, the first agent comprises biotin conjugated to thefirst complementary sequence.

The first agent can further comprise a first reporter-labeled conjugatethat binds to the first recognition element, as shown in FIG. 4. Thefirst reporter-labeled conjugate may be an avidin, an antibody, anaptamer, combinations thereof, or any known acceptable conjugate thatbinds the recognition element. In some embodiments, the firstreporter-labeled conjugate can be labeled with a first reporter. In someembodiments, the first reporter is a fluorophore.

In some embodiments, the first agent can further comprise a firstconjugate antibody, as shown in FIG. 4. In illustrative embodiments, thefirst conjugate antibody binds to the first reporter-labeled conjugate.In some embodiments, the first conjugate antibody comprises arecognition element. In some embodiments, the recognition element of thefirst conjugate antibody can be the same as the first recognitionelement. In some examples, the first conjugate antibody can be labeledwith biotin.

In some embodiments, the first reporter-labeled conjugate binds therecognition element conjugated to the first complementary sequence, thefirst conjugate antibody, or both the recognition element conjugated tothe first complementary sequence and the first conjugate antibody, asshown in FIG. 4. In some embodiments, the first reporter-labeledconjugate binds both the recognition element conjugated to the firstcomplementary sequence and the first conjugate antibody, each of thefirst reporter labeled conjugates comprises the same first reporter.

The first reporter may be a fluorophore, an enzymatic tag such as anHRP, a radioisotope, a combination thereof, or any suitable reportertypically used in biochemical assays, as shown in FIG. 4. In someembodiments, the fluorophore can have an emission peak between about 640nm and about 680 nm. In some embodiments, the fluorophore isallophycocyanin.

After or during the step of hybridization, a second agent that binds thefirst nucleic acid sequence of the amplified product compositions can beintroduced, as shown in FIG. 4. In some embodiments, the second agentcomprises a complementary sequence to a portion of the second targetsequence (e.g. the second nucleic acid sequence).

In some embodiments, the second agent comprises the second complementarysequence and a second recognition element conjugated to the secondcomplementary sequence, as shown in FIG. 4. Illustrative examples ofsecond recognition elements include fluorophores, biotin, peptide tags,combinations thereof, or any known acceptable recognition element knownin the art. In some embodiments, the second agent comprises afluorophore conjugated to the second complementary sequence. In someembodiments, the fluorophore can be FAM.

The second agent can further comprise a second reporter-labeledconjugate that binds to the second recognition element, as shown in FIG.4. The second reporter-labeled conjugate may comprise an avidin, anantibody, an aptamer, combinations thereof, or any known acceptableconjugate that binds the recognition element. In some embodiments, thesecond reporter-labeled conjugate comprises an antibody. In someembodiments, the second reporter-labeled conjugate can be labeled with asecond reporter. In some embodiments, the second reporter is afluorophore.

In some embodiments, the second agent can further comprise a secondconjugate antibody, as shown in FIG. 4. In illustrative embodiments, thesecond conjugate antibody binds to the second reporter-labeledconjugate. In some embodiments, the second conjugate antibody comprisesa recognition element. In some examples, the recognition element of thesecond conjugate antibody can be the same as the second recognitionelement. In some examples, the second conjugate antibody can be labeledwith FAM.

In some embodiments, the second reporter-labeled conjugate binds therecognition element conjugated to the second complementary sequence, thesecond conjugate antibody, or both the recognition element conjugated tothe second complementary sequence and the second conjugate antibody, asshown in FIG. 4. In some embodiments, the second reporter-labeledconjugate binds both the recognition element conjugated to the secondcomplementary sequence and the second conjugate antibody, each of thesecond reporter labeled conjugates comprises the same second reporter.

The second reporter may be a fluorophore, an enzymatic tag such as anHRP, a radioisotope, a combination thereof, or any suitable reportertypically used in biochemical assays, as shown in FIG. 4. In someembodiments, the fluorophore can have an emission peak between about 560nm and about 600 nm. In some embodiments, the fluorophore isphycoerythin.

It will be appreciated that in some embodiments, the first agent can beconfigured to bind the amplified product compositions derived from thefirst channel and the second agent can be configured to bind theamplified product compositions of the second channel. It should beequally appreciated that in some embodiments, the first agent can beconfigured to bind the amplified product compositions derived from thesecond channel and the second agent can be configured to bind theamplified product compositions of the first channel. Accordingly, insome embodiments, the reporters (e.g. the fluorophore(s)) of the firstagent are different than the reporters (e.g. the fluorophore(s)) of thesecond agent, as shown in FIG. 4.

In some embodiments, a set of probes (e.g., a set of probes targeting atest chromosome, e.g., Chromosome 21), may target different regions of atarget polynucleotide, yet each probe within the set has the sameuniversal primer-binding sites. In some cases, each probe has the sameprobe-binding site. In some cases, two or more probes in the reactionmay have different probe-binding sites. In some cases, the probes addedto such reactions are conjugated to the identical signal agent (e.g.,fluorophores of the same color). In some cases, different signal agents(e.g., two different colors) are conjugated to one or more probes.

The oligonucleotide probe may also comprise a sequence that iscomplementary to a probe attached to a marker, such as a dye orfluorescent dye (e.g., TaqMan probe). In some cases, the TaqMan probe isbound to one type of dye (e.g., FAM, VIC, TAMRA, ROX). In other cases,there are more than one TaqMan probe sites on the oligonucleotide, witheach site capable of binding to a different TaqMan probe (e.g., a TaqManprobe with a different type of dye). There may also be multiple TaqManprobe sites with the same sequence of the oligonucleotide probedescribed herein. Often, the TaqMan probe may bind only to a site on theoligonucleotide probe described herein, and not to genomic DNA, but insome cases a TaqMan probe may bind genomic DNA.

Analysis

In some embodiments, the disclosed methods (as well as relatingcompositions, systems, instruments and software) include a step ofanalyzing the data obtained from the array to analyze the properties ofthe nucleic acid sample (or derivative thereof) that is applied to thearray. In some embodiments, the nucleic acid sample includes a mixednucleic acid population containing a major subpopulation and a minorsubpopulation.

In some embodiments, the disclosed methods can include detecting one ormore signals from the oligonucleotide array using a detector.

Optionally, the detecting includes detecting a signal (“first signal” or“A signal”) indicating the presence or absence of a first nucleotidevariant. The first nucleotide variant optionally corresponds to a firstallelic variant.

Optionally, the detecting includes detecting a signal (“second signal”or “B signal”) indicating the presence or absence of a second nucleotidevariant. The second nucleotide variant optionally corresponds to asecond allelic variant.

In some embodiments, the disclosed methods can include determining thecopy number of the first chromosomal region in the minor subpopulationusing the first signal and the second signal.

In some embodiments, the disclosed methods can include determining thecopy number of the first chromosomal region in the major subpopulationusing the first signal and the second signal.

In some embodiments, the disclosed methods can include determining thegenotype of the polymorphic site for the minor subpopulation using thefirst signal and the second signal.

In some embodiments, the disclosed methods can include determining thegenotype of the polymorphic site for the major subpopulation using thefirst signal and the second signal.

In some embodiments, the disclosed methods can include determining therelative amounts of the major subpopulation and the minor subpopulationin the mixed nucleic acid population using the first signal and thesecond signal.

In some embodiments, the methods can include calculating the ratio ofthe first signal to the second signal, or the log ratio of the signals.

In some embodiments, the methods include analyzing the A signal and theB signal from an array feature configured to hybridize to a targetnucleic acid containing a polymorphic site, and using the A signal andthe B signal to determine both the genotype of the polymorphic sitewithin the major and the minor subpopulations, as well as the copynumber (or relative copy number) of the polymorphic site within themajor and minor subpopulations.

Kits

Kits for performing the disclosed methods are also disclosed. The kitsmay comprise pools of molecular inversion probes designed foramplification of a plurality of target sequences. The target sequencesare selected so that they each contain a polymorphic site of interest.The molecular inversion probes may be pooled into containers thatcontain 2 or more different sequence capture probes. The kit may furthercomprise adaptors, universal primers, dNTPs, ligase, buffer, andpolymerase.

The kits may be used to amplify a collection of target sequencesAmplification may be by fragmentation of the sample, ligation of anadaptor to the fragments, hybridization of capture probes to theadaptor-ligated fragments, extension of the capture probe, andamplification of the extended capture probes using a pair of universalprimers.

The kits may also include a computer system for reading and analyzingmircoarray data. In addition, the kits may include a microarray chip forhybridizing and labeling the target sequences.

Applications

The methods and systems described herein can be used to detect geneticabnormalities of numerous types that are indicative of the presence of adisease or the possibility of developing a disease. For example, asdescribed herein, the present disclosure can be useful for detectingcopy number variants in a maternal sample that includes a majorsubpopulation and a minor subpopulation, wherein the major and minorsubpopulations each include a target sequence located in a firstchromosomal region and containing a polymorphic site. In someembodiments, the major population is maternal DNA. In some embodiments,the minor population is fetal DNA. In some embodiments, the fetal DNA isno greater than 15% of total DNA in the nucleic acid sample, or nogreater than 10% of total DNA in the nucleic acid sample, or no greaterthan 5% of total DNA in the nucleic acid sample. In some embodiments,the major subpopulation is genotyped according to the methods describedherein. In some embodiments, the minor subpopulation is genotypedaccording to the methods described herein.

In some embodiments, a sample includes a mixed nucleic acid populationfrom different subpopulations (e.g., major and minor subpopulations). Inone embodiment, a sample contains a mixture of maternal nucleic acids (amajor subpopulation) and fetal nucleic acids (a minor subpopulation.) Inone embodiments, the nucleic acids from each subpopulation are cell-freeDNA. In some embodiments, the amount of the fetal DNA in a sample rangesfrom about 1% to about 50% of the total amount of DNA in the sample. Insome embodiments, the amount of the fetal DNA in the sample is about 1%,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45% or about 50% of the total amount of DNA in thesample, or any intervening amount of the foregoing. In some embodiments,the amount of the fetal DNA in the sample is no greater than about 1%,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45% or about 50% of the total amount of DNA in thesample, or any intervening amount of the foregoing. In some embodiments,the amount of the fetal DNA in the sample is more or no less than about1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45% or about 50% of the total amount of DNAin the sample, or any intervening amount of the foregoing.

In some embodiments, the mixed nucleic acid population in a sample thatcan be processed according to various methods disclosed herein includescell-free DNA from major and minor sources. In some embodiments, themixed nucleic acid population is circulating DNA isolated from wholeblood, plasma, serum or some other bodily fluid. In some embodiments,the mixed nucleic acid population includes maternal and fetal cell-freeDNA. In some embodiments, the amount of mixed nucleic acid population ina sample is in the range from one or more nanograms (ngs) to about oneor more milligrams (mgs). In some embodiments, the amount mixed nucleicacid population is about 1 ng, about 3 ngs, about 5 ngs, about 10 ngs,about 15 ngs, about 30 ngs, about 40 ngs, about 50 ngs, about 100 ngs,about 150 ngs, about 300 ngs, about 400 ngs, about 500 ngs, about 1 mg,about 3 mgs, about 5 mgs or more, or any intervening amount of theforegoing. In some embodiments, the amount of the mixed nucleic acidpopulation used is no greater than about 50 ngs, about 40 ngs, about 30ngs, about 15 ngs, about 10 ngs, about 5 ngs, about 3 ngs or about 1 ng.In some embodiments, the amount mixed nucleic acid population is aboutor less than about 50 ngs, about 40 ngs, about 30 ngs, about 15 ngs,about 10 ngs, about 5 ngs, about 3 ngs or about 1 ng.

In some embodiments, a sample that is processed according to variousmethods disclosed herein includes a mixed nucleic acid populationderived from one or more of whole blood, plasma, serum, urine, stool orsaliva. In some embodiments, a mixed nucleic acid population can bederived from blood. In some embodiments, blood, e.g., whole blood can befurther processed to provide plasma and/or serum from which a mixednucleic acid population for a sample is prepared.

In some embodiments, the disclosed methods (as well as relatedcompositions, kits and systems) are useful in detecting genetic changesin small amounts of whole blood, plasma, serum or other bodily fluid.For example, the amount of bodily fluid (e.g., whole blood, plasma,serum or saliva) that is used to prepare a mixed nucleic acid populationof a sample can be in the range of about 0.1 to several milliliters(mis). In some embodiments, the amount of whole blood, plasma, serum orother bodily fluid that is used to prepare a mixed nucleic acidpopulation is about 0.1 ml, about 0.25 ml, about 0.5 ml, about 0.75 ml,about 1 ml, about 1.5 ml, about 2 mls, about 2.5 mls, about 3 mls, about3.5 mls, about 4 mls, about 4.5 mls, about 5 mls about 5.5 mls, about 6mls, about 6.5 mls, about 7 mls, about 7.5 mls, about 8 mls, about 8.5mls, about 9 mls, about 9.5 mls, or about 10 mls, or any interveningvolumes of the foregoing.

In some embodiments where whole blood is used to provide a mixed nucleicacid population of a sample, the amount of blood is about or less than0.1 ml, 0.25 ml, about 0.5 ml, about 0.75 ml, about 1 ml, about 1.5 ml,about 2 mls, about 2.5 mls or about 3 mls. In some embodiments, theamount of blood is no greater than about 0.25 ml, about 0.5 ml, about0.75 ml, about 1 ml, about 1.5 ml, about 2 mls, about 2.5 mls or about 3mls.

In some embodiments where plasma or serum is used to provide a mixednucleic acid population of a sample, the amount of plasma or serum isabout or less than 0.1 ml, 0.25 ml, about 0.5 ml, about 0.75 ml, about 1ml, about 1.5 ml, about 2 mls, about 2.5 mls or about 3 mls. In someembodiments, the amount of plasma or serum is no greater than about 0.25ml, about 0.5 ml, about 0.75 ml, about 1 ml, about 1.5 ml, about 2 mls,about 2.5 mls or about 3 mls.

The methods and systems described herein can also be used to detectcirculating tumor cells from a biological sample, e.g. blood thatcontains a major subpopulation and a minor subpopulation, wherein themajor and minor subpopulations each include a target sequence located ina first chromosomal region and containing a polymorphic site. In someembodiments, the minor subpopulation can be genotyped to identify aknown genetic marker for cancer, such as a SNP, a chromosomal inversion,a chromosomal deletion, a chromosomal insertion, and the like. It willbe appreciated that numerous markers for cancer are known in the art.

EXAMPLES Example 1: Annealing

Annealing was performed as generally described for the Oncoscan™ FFPEAssay kit (catalog #902293) available from Thermo Fisher.

Briefly, an assay microwell plate of 96 samples was prepared on ice. 10μL of DNA was added to each well. The DNA sample may be an analyticalgDNA sample (sheared to a median length of 170 bp); an analyticalmixture of gDNA mixed with trisomy gDNA at 0, about 5%, or about 10%trisomy to analytical gDNA (sheared to a median length of 170 bp); orclinical cell-free DNA (cfDNA) purified from 10-20 mL maternal bloodsamples by MagMAX (available from Thermo Fisher) extraction kit methods.

An Anneal Master Mix (AMM) was prepared by mixing Buffer A of theOncoscan™ FFPE Assay kit with a MIP probe mix containing about 48,000MIPs from the OncoScan™ library. About 2.24 μL of AMM was added to eachDNA sample and the reagents were mixed, vortexed, and centrifuged.

The microwell plate was placed in a thermocycler and incubated overnightaccording to the Oncoscan™ FFPE Assay protocol.

Example 2: Gap Filling and Channel Split

The gap filling was performed as generally described for the Oncoscan™FFPE Assay.

Briefly, Buffer A, dNTPs, and the Cleavage Buffer were thawed on ice.

SAP recombinant enzyme was mixed with Buffer A and the Gap Fill EnzymeMix. 2 μL of the prepared mixture was added to the microwell plate fromExample 1. The contents of wells were then split equally into two newmicrowell plates to create two channels.

The microwell plates were placed in a thermocycler and incubated for 11minutes using the Gap Fill program as described in the Oncoscan™ manual.

Example 3: dNTP Addition

2.4 μL of an ATP/TTP mix or a GTP/CTP were added to wells containing theDNA as described in Example 2. The microwell plates were placed back ina thermocycler to complete the Gap-Fill program.

Example 4: Exonuclease Treatment

An Exo Master Mix (EMM) was prepared by mixing the Exo Mix from theOncoscan™ kit with glycerol and the wells were treated as described inthe Oncoscan™ FFPE Assay.

Briefly, 2 μL of EMM was added and mixed with the solutions in themicrowell plate from Example 3. The microwell plates were placed in athermocycler and the program according to the Oncoscan™ FFPE Assay wascontinued.

Example 5: Cleavage and PCR

A Cleavage Master Mix (CMM) was prepared by mixing the Cleavage Bufferand Cleavage Enzyme according to the Oncoscan™ FFPE Assay. PCR mixeswere prepared by mixing a complement mix (either A/T or C/G) withTitanium Taq (available from ClonTech).

15.0 μL of CMM was added to the wells of the microwell plate fromExample 4 and mixed.

15.0 μL of the PCR mixes were added to the appropriate wells and mixed.

The microwell plates were placed in a thermocycler and incubatedaccording to the Cleavage-PCR program as described in the Oncoscan™ FFPEAssay.

Example 6: Digestion

The digestion step was performed according to the Oncoscan™ FFPE Assay.

Briefly, Buffer B was thawed on ice. A HaeIII Master Mix (H3MM) wasprepared by mixing Buffer B with HaeIII and the ExoI enzyme according tothe Oncoscan™ FFPE Assay.

40 μL of H3MM was added to each sample well on a new microwell plate. Toeach filled well, 10 μL of an A/T product was mixed with 10 μL of a C/Gproduct and mixed.

The plate was placed in a thermocycler and incubated using the HaeIIIDigest program according to the Oncoscan™ FFPE Assay.

Example 7: Denaturation and Hybridization

The denaturation and hybridization were performed according to and withreagents from the Axiom 2.0 reagent kit (catalog #901758) available fromThermo Fisher.

Briefly, the Hybe Mix was thawed on ice and then 82.3 μL/well waspipetted into a microwell plate.

36 μL of the digested product from Example 6 was added to each wellcontaining the Hybe Mix. The plate was incubated for 25 minutes at roomtemperature. The microwell plate was then incubated in a thermocycler at95° C. for 10 minutes, then 49° C. for at least 3 minutes.

About 100 μL of the denatured product from each well was added to theHybe tray from the Axiom 2.0 kit and the plate was placed in aGeneTitan™Multi-Channel (GTMC) instrument and incubated for 23.5 hours.

Example 8: Washing, Fixing, and Staining

The Hybe tray was washed and stained generally according to the Axiom2.0 manual.

Briefly, a holding tray was prepared by adding 150 μL of the Axiomholding buffer into each well of a microwell plate. Astabilization/fixing solution was prepared according to the Axiom 2.0manual and 150 μL of the solution was added into each well of amicrowell plate.

A first stain mix was prepared according to the Axiom 2.0 manual andmodified by using a polyclonal antibody and 105 μL of the solution wasadded into each well of two microwell plates.

A second stain mix was prepared according to the Axiom 2.0 manual andmodified by using a polyclonal antibody and 105 μL of the solution wasadded into each well of a microwell plate.

The trays were added to the GTMC instrument. The GTMC instrumentperformed the washing, staining, fixing, and holding-filling accordingto the Axiom 2.0 manual.

Example 9: Collecting the Data

The stained tray from Example 8 was imaged according to the Axiom 2.0protocol. The data was collected and analyzed.

What is claimed is:
 1. A method for analyzing a mixed nucleic acidsample obtained from an organism, comprising: obtaining or deriving froman organism a nucleic acid sample containing a mixed nucleic acidpopulation that includes a major subpopulation and a minorsubpopulation, wherein the major and minor subpopulations each include atarget sequence located in a first nucleic acid locus and containing apolymorphic site, wherein the polymorphic site can include combinationsof a first nucleotide variant and a second nucleotide variant;genotyping the polymorphic site, wherein the genotyping includes: (a)contacting the nucleic acid sample with a pool of linear molecularinversion probes to provide an annealing mixture, wherein portions ofthe linear molecular inversion probes hybridize to the target sequence;(b) dividing the annealing mixture into a first channel composition anda second channel composition; (c) adding a mixture of deoxynucleotidesto each of the first and second channel composition, wherein the mixtureof deoxynucleotides added to the first channel composition is differentfrom the mixture of deoxynucleotides added to the second channelcomposition; (d) contacting the first and second channel compositionswith a ligase to form first and second circularized probe compositionsfrom the linear molecular inversion probes: (e) cleaving the first andsecond circularized probe compositions to form first and secondlinearized probe compositions; (f) combining the first and secondlinearized probe compositions; (g) hybridizing the first and secondlinearized probe compositions to an oligonucleotide probe of anoligonucleotide array; and (h) detecting from the oligonucleotide array,using a detector, a first signal indicating the presence or absence ofthe first nucleotide variant (“A signal”) corresponding to a firstallelic variant and a second signal indicating the presence or absenceof the second nucleotide variant (“B signal”) corresponding to a secondallelic variant.
 2. The method of claim 1, further including determininga copy number of the first nucleic acid locus in the minorsubpopulation, the major subpopulation, or both the major and minorsubpopulations, using the first signal and the second signal.
 3. Themethod of claim 1, further including determining a copy number of achromosome containing the first nucleic acid locus in the majorsubpopulation, the minor subpopulation or both the major and minorsubpopulations, using the first signal and the second signal.
 4. Themethod of claim 1, further including determining a genotype of thepolymorphic site for the minor subpopulation and/or the majorsubpopulation using the first signal and the second signal.
 5. Themethod of claim 1, further including determining relative amounts of themajor subpopulation and the minor subpopulation in the mixed nucleicacid population using the first signal and the second signal.
 6. Themethod of claim 1, wherein the major subpopulation and the minorsubpopulation originate from different sources in the organism.
 7. Themethod of claim 1, wherein the mixed nucleic acid population includescell-free DNA.
 8. The method of claim 7, wherein the cell-free DNA isobtained or derived from blood, plasma, serum, urine, stool or saliva ofthe organism.
 9. The method of claim 1, wherein the organism includes atumor, the major subpopulation includes or is derived from normal tissueand the minor subpopulation includes or is derived from the tumor. 10.The method of claim 1, wherein the organism is a pregnant female, themixed nucleic acid population is cell-free DNA obtained from blood ofthe pregnant female, the major subpopulation includes or is derived frommaternal nucleic acid and the minor subpopulation includes or is derivedfrom fetal nucleic acid.
 11. The method of claim 1, wherein thepolymorphic site includes a bi-allelic SNP, the first nucleotide variantis a first allelic variant of the SNP (“A allele”) and the secondnucleotide variant is a second allelic variant of the SNP (“B allele”).12. The method of claim 1, further comprising multiplex genotyping ofmultiple polymorphic sites, wherein at least 50% of the pool of linearmolecular inversion probes are configured to target sequences of themixed nucleic acid sample from chromosomes 1, 5, 13, 18, 21, X, and Y.13. The method of claim 1, wherein the mixed nucleic acid samplecomprises DNA fragment copies, and wherein a ratio of a total number ofthe linear molecular inversion probes to a total number of DNA fragmentcopies is at least 15,000:1.
 14. The method of claim 1, wherein the stepof genotyping further includes amplifying the first and secondlinearized probe compositions prior to hybridizing the first and secondlinearized probe compositions to the oligonucleotide probe of theoligonucleotide array.
 15. The method of claim 1, wherein the step ofdetecting further includes, after the step of hybridizing the first andsecond linearized probe compositions to the oligonucleotide probe of theoligonucleotide array, labeling the first and second linearized probecompositions with a first agent that binds to the first linearized probecomposition and a second agent that binds to the second linearized probecomposition.
 16. The method of claim 15, wherein the first agentcomprises a complementary sequence to a portion of the first linearizedprobe composition.
 17. The method of claim 15, wherein the second agentcomprises a complementary sequence to a portion of the second linearizedprobe composition.
 18. The method of claim 14, wherein the genotyping ofthe polymorphic site further comprises cleaving the first and secondlinearized probe compositions at one or more restriction enzymerecognition sites after amplifying of the first and second linearizedprobe compositions.